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1.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(8): 988-993, 2020 Aug 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33053543

RESUMO

OBJECTIVES: Reflection confocal microscopy (RCM) is commonly used to assist in the diagnosis of skin diseases. RCM has been found to be useful in the diagnosis of genital warts. The characteristics of genital warts under RCM are not specific. This study aimed to face the challenge of diagnosing genital warts with RCM. METHODS: We retrospectively studied 161 patients with clinical diagnosis of genital warts who underwent RCM examination. Histopathological examination was performed for 75 of these patients. The results of histological diagnosis and RCM diagnosis were compared and analyzed. RESULTS: According to the RCM images, 102 of the 161 (63.35%) patients were diagnosed as genital warts. By comparison with histological results, the misdiagnosis rate of the 75 biopsy cases was 35.48% and the missed diagnosis rate was 20.45%.The diseases of misdiagnosis and missed diagnosis mainly included bowenoid papulosis, pseudocondylona of vulvae, squamous cell carcinoma in situ, molluscum contagiosum, and lichen planus. We further found that the RCM characteristics of genital warts conformed by histology were koilocytes, which mainly appeared in the granular layer or the spinous layer, larger than normal skin keratinocytes, and the cytoplasm with low refractive index. CONCLUSIONS: Due to the commonality of the microscopic characteristics of genital warts and related diseases, some cases are easy to be misdiagnosed or missed diagnosed. Diagnostic koilocytes could be an important basis for the diagnosis of genital warts.


Assuntos
Carcinoma de Células Escamosas , Condiloma Acuminado , Neoplasias Cutâneas , Condiloma Acuminado/diagnóstico , Condiloma Acuminado/diagnóstico por imagem , Humanos , Microscopia Confocal , Estudos Retrospectivos
2.
Zhonghua Yan Ke Za Zhi ; 56(10): 754-760, 2020 Oct 11.
Artigo em Chinês | MEDLINE | ID: mdl-33059418

RESUMO

Objective: To study the diabetic keratopathy in type 2 diabetes patients with retinopathy by in vivo laser confocal microscopy. Methods: This was a case-control study. Ninety type 2 diabetes patients were involved in this study from May 2015 to December 2019 in Qingdao Eye Hospital. According to the diabetic retinopathy clinical stage, these patients were divided into the non-proliferative diabetic retinopathy (NPDR) group (30 cases), early stage proliferative diabetic retinopathy (PDR) group (30 cases) and intermediate to late stage PDR group (30 cases). Thirty non-diabetic healthy volunteers were included in the control group. The central cornea was observed with an in vivo laser confocal microscope. The corneal nerve fiber density, nerve fiber length, nerve branch density, and nerve fiber tortuosity were compared between groups. The corneal Langerhans cells, epithelial cells, stromal cells and endothelial cells were also compared. Results: There were more nerve fibers and branches in the control group than the other three diabetic groups. The nerve fiber length in the control group, NPDR group, early stage PDR group and intermediate to late stage PDR group was (21.55±2.57), (14.73±1.56), (11.23±1.40) and (8.02±1.33) mm/mm2, respectively, and there were statistically significant differences between the groups (F=316.17, P=0.00). In the nerve fiber density, nerve branch density and curvature, there were statistically significant differences between the groups (F=345.72, 479.46, 167.00, all P=0.00). The basal cell density in the control group, NPDR group, and two PDR groups was (5 761±303), (5 336±367), (4 146±379) and (3 658±365) cells/mm2, respectively, and there were statistically significant differences between the groups (F=234.94, P=0.00). The anterior stromal cell density in the four groups was (836±30), (727±57), (544±59) and (360±47) cells/mm2, respectively, and there were statistically significant differences between the groups (F=535.08, P=0.00). The hexagonal endothelium cell rate in the four groups was 62.0%±5.5%, 51.1%±3.7%, 40.2%±4.0% and 27.8%±3.9%, respectively, and the Langerhans cell density was (1.5±0.6), (4.2±1.3), (6.8±2.1) and (10.9±2.1) cells/mm2, respectively; there were statistically significant differences between the groups (F=342.28, 179.78, all P=0.00). There was no statistically significant difference between the groups in the corneal endothelial cell density (F=1.58, P=0.20). Conclusions: In type 2 diabetes patients with diabetic retinopathy, the corneal nerve fiber and branch density can be significantly reduced, and the density of the hexagonal corneal endothelial cells, epithelial basal cells and anterior stromal cells can also decrease. Langerhans cells may be involved in the development diabetic keratopathy. (Chin J Ophthalmol, 2020, 56: 754-760).


Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/diagnóstico por imagem , Células Endoteliais , Humanos , Microscopia Confocal
3.
Nat Commun ; 11(1): 4655, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938918

RESUMO

Purely organic room-temperature phosphorescence has attracted attention for bioimaging but can be quenched in aqueous systems. Here we report a water-soluble ultralong organic room-temperature phosphorescent supramolecular polymer by combining cucurbit[n]uril (CB[7], CB[8]) and hyaluronic acid (HA) as a tumor-targeting ligand conjugated to a 4-(4-bromophenyl)pyridin-1-ium bromide (BrBP) phosphor. The result shows that CB[7] mediated pseudorotaxane polymer CB[7]/HA-BrBP changes from small spherical aggregates to a linear array, whereas complexation with CB[8] results in biaxial pseudorotaxane polymer CB[8]/HA-BrBP which transforms to relatively large aggregates. Owing to the more stable 1:2 inclusion complex between CB[8] and BrBP and the multiple hydrogen bonds, this supramolecular polymer has ultralong purely organic RTP lifetime in water up to 4.33 ms with a quantum yield of 7.58%. Benefiting from the targeting property of HA, this supramolecular polymer is successfully applied for cancer cell targeted phosphorescence imaging of mitochondria.


Assuntos
Mitocôndrias/efeitos dos fármacos , Polímeros/química , Células A549 , Células HEK293 , Humanos , Ácido Hialurônico/química , Ligação de Hidrogênio , Medições Luminescentes , Microscopia Confocal , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Polímeros/metabolismo , Taxoides/química , Temperatura
4.
Nat Commun ; 11(1): 4482, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901011

RESUMO

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.


Assuntos
DNA/metabolismo , Sondas Moleculares/química , Nanopartículas/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico Ativo , Técnicas Biossensoriais/métodos , Química Click , Citometria de Fluxo , Corantes Fluorescentes , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Células NIH 3T3
5.
Life Sci ; 259: 118390, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896556

RESUMO

AIMS: This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto-maternal interface. MAIN METHODS: The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. KEY FINDINGS: ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in all cell types. SIGNIFICANCE: ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Troca Materno-Fetal , Receptor 4 Toll-Like/metabolismo , Western Blotting , Células Cultivadas , Córion/metabolismo , Decídua/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Placenta/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
PLoS One ; 15(8): e0233247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857759

RESUMO

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.


Assuntos
Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Solubilidade
7.
Nat Commun ; 11(1): 4107, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796836

RESUMO

Foamy macrophages, which have prominent lipid droplets (LDs), are found in a variety of disease states. Toll-like receptor agonists drive triacylglycerol (TG)-rich LD development in macrophages. Here we explore the basis and significance of this process. Our findings indicate that LD development is the result of metabolic commitment to TG synthesis on a background of decreased fatty acid oxidation. TG synthesis is essential for optimal inflammatory macrophage activation as its inhibition, which prevents LD development, has marked effects on the production of inflammatory mediators, including IL-1ß, IL-6 and PGE2, and on phagocytic capacity. The failure of inflammatory macrophages to make PGE2 when TG-synthesis is inhibited is critical for this phenotype, as addition of exogenous PGE2 is able to reverse the anti-inflammatory effects of TG synthesis inhibition. These findings place LDs in a position of central importance in inflammatory macrophage activation.


Assuntos
Inflamação/metabolismo , Lipidômica/métodos , Triglicerídeos/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Glucose/metabolismo , Glicerol/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica , Palmitatos/metabolismo , Análise de Sequência de RNA
8.
Int J Nanomedicine ; 15: 5671-5685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32821096

RESUMO

Aim: The aim of the current work was to develop vardenafil hydrochloride (VRD)-loaded ethosome-derived invasomes as a possible transdermal system which could be used for patients suffering from pulmonary arterial hypertension. Methods: VRD-loaded ethosomes were developed at three concentrations of phosphatidylcholine (5, 10 and 15 mg/mL) and three percentages of ethanol (20%, 30% and 40%, v/v). The best achieved VRD-loaded ethosomes (ETH9) were optimized to invasomes via incorporation of terpenes (limonene, cineole and a 1:1 mixture) at three concentrations (0.5%, 1% and 2%, v/v). All systems were evaluated for vesicle size, zeta potential, drug entrapment efficiency (EE%), cumulative drug permeated percentages after 0.5hrs (Q0.5h) and 12hrs (Q12h) and steady-state flux (Jss). The optimized system (ETH9-INV8) was further characterized for morphology, histopathology and confocal laser scanning microscopy (CLSM). Physiologically based pharmacokinetic (PBPK) modeling was employed to estimate VRD pharmacokinetic parameters from the optimized transdermal system and an oral aqueous drug dispersion, in adults and geriatrics. Results: The optimized invasomal system (ETH9-INV8) was characterized with spherical vesicles (159.9 nm) possessing negative zeta potential (-20.3 mV), promising EE% (81.3%), low Q0.5h (25.4%), high Q12h (85.3%) and the largest steady-state flux (6.4 µg.cm-2h-1). Following a leave-on period of 12hrs in rats, it showed minor histopathologic changes. CLSM studies proved its ability to deeply permeate rat skin. Lower Cmax values, delayed Tmax estimates and greater AUC0-24h folds in adults and geriatrics (≈ 2.18 and 1.69, respectively) were estimated following the transdermal application of ETH9-INV8 system. Conclusion: ETH9-INV8 is a promising transdermal system for VRD.


Assuntos
Sistemas de Liberação de Medicamentos , Etanol/química , Geriatria , Modelos Biológicos , Dicloridrato de Vardenafila/administração & dosagem , Dicloridrato de Vardenafila/farmacocinética , Administração Cutânea , Animais , Lipossomos , Masculino , Microscopia Confocal , Tamanho da Partícula , Permeabilidade , Ratos Wistar , Absorção Cutânea , Eletricidade Estática
9.
PLoS Comput Biol ; 16(8): e1008049, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822341

RESUMO

Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.


Assuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Morfogênese/fisiologia , Algoritmos , Animais , Divisão Celular/fisiologia , Mineração de Dados/métodos , Drosophila/citologia , Drosophila/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Feminino , Masculino , Microscopia Confocal , Fatores de Tempo
10.
Nat Commun ; 11(1): 3894, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753597

RESUMO

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17-29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717-29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Amiloide/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/metabolismo , Benzotiazóis , Catelicidinas/farmacologia , Cristalografia por Raios X , Gorilla gorilla , Humanos , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Staphylococcus hominis/efeitos dos fármacos , Difração de Raios X
11.
Methods Mol Biol ; 2203: 241-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833217

RESUMO

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.


Assuntos
Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Microscopia Confocal/métodos , Linhagem Celular , Coronavirus/isolamento & purificação , Meios de Cultura/química , Endocitose , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Ácidos Tri-Iodobenzoicos/química , Internalização do Vírus
12.
AAPS PharmSciTech ; 21(6): 227, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32767025

RESUMO

Streptococcus mutans (S. mutans) is the principal etiologic agent in the occurrence of human dental caries and the formation of biofilms on the surface of teeth. Tea tree oil (TTO) has been demonstrated to exhibit a wide range of pharmacological actions that can effectively inhibit the activity of bacteria. In this context, we evaluated the in vitro antimicrobial effects of TTO on S. mutans both during planktonic growth and in biofilms compared with 0.2% CHX. We determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the microdilution method, the bacteriostatic rate using an MTT assay, and the antimicrobial time using a time-kill assay. Then, we explored the effects of TTO on acid production and cell integrity. Furthermore, the effects of TTO on the biomass and bacterial activity of S. mutans biofilms were studied. Finally, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to investigate the structure and activity of biofilms. The MIC and MBC values were 0.125% and 0.25%, and the bacterial inhibition rate was concentration dependent. TTO can effectively inhibit bacterial acid production and destroy the integrity of the cell membrane. Electron micrographs revealed a reduction in bacterial aggregation, inhibited biofilm formation, and reduced biofilm thickness. The effect of TTO was the same as that of 0.2% CHX at a specific concentration. In summary, we suggest that TTO is a potential anticariogenic agent that can be used against S. mutans.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Óleo de Melaleuca/farmacologia , Cárie Dentária/microbiologia , Humanos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Microscopia Confocal , Microscopia Eletrônica de Varredura , Streptococcus mutans/efeitos dos fármacos
13.
PLoS One ; 15(8): e0238104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822415

RESUMO

PURPOSE: To delineate responses of optic nerve head astrocytes to sustained intraocular pressure (IOP) elevation in mice. METHODS: We elevated IOP for 1 day to 6 weeks by intracameral microbead injection in 4 strains of mice. Astrocyte alterations were studied by transmission electron microscopy (TEM) including immunogold molecular localization, and by laser scanning microscopy (LSM) with immunofluorescence for integrin ß1, α-dystroglycan, and glial fibrillary acidic protein (GFAP). Astrocyte proliferation and apoptosis were quantified by Ki67 and TUNEL labeling, respectively. RESULTS: Astrocytes in normal optic nerve head expressed integrin ß1 and α-dystroglycan by LSM and TEM immunogold labeling at electron dense junctional complexes that were found only on cell membrane zones bordering their basement membranes (BM) at the peripapillary sclera (PPS) and optic nerve head capillaries. At 1-3 days after IOP elevation, abnormal extracellular spaces appeared between astrocytes near PPS, and axonal vesical and mitochondrial accumulation indicated axonal transport blockade. By 1 week, abnormal spaces increased, new collagen formation occurred, and astrocytes separated from their BM, leaving cell membrane fragments. Electron dense junctional complexes separated or were absent at the BM. Astrocyte proliferation was modest during the first week, while only occasional apoptotic astrocytes were observed by TEM and TUNEL. CONCLUSIONS: Astrocytes normally exhibit junctions with their BM which are disrupted by extended IOP elevation. Responses include reorientation of cell processes, new collagen formation, and cell proliferation.


Assuntos
Astrócitos/fisiologia , Glaucoma/patologia , Nervo Óptico/fisiologia , Animais , Apoptose , Astrócitos/citologia , Astrócitos/patologia , Proliferação de Células , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nervo Óptico/citologia , Nervo Óptico/patologia
14.
PLoS One ; 15(8): e0237795, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833989

RESUMO

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.


Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Buffy Coat/citologia , Diferenciação Celular , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Técnicas de Cocultura , Voluntários Saudáveis , Humanos , Microscopia Intravital , Macrófagos/citologia , Macrófagos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Monócitos/fisiologia , Cultura Primária de Células
15.
Nat Commun ; 11(1): 4218, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839452

RESUMO

Exposure to social stress and dysregulated serotonergic neurotransmission have both been implicated in the etiology of psychiatric disorders. However, the serotonergic circuit involved in stress vulnerability is still unknown. Here, we explored whether a serotonergic input from the dorsal raphe (DR) to ventral tegmental area (VTA) influences vulnerability to social stress. We identified a distinct, anatomically and functionally defined serotonergic subpopulation in the DR that projects to the VTA (5-HTDR→VTA neurons). Moreover, we found that susceptibility to social stress decreased the firing activity of 5-HTDR→VTA neurons. Importantly, the bidirectional manipulation of 5-HTDR→VTA neurons could modulate susceptibility to social stress. Our findings reveal that the activity of 5-HTDR→VTA neurons may be an essential factor in determining individual levels of susceptibility to social stress and suggest that targeting specific serotonergic circuits may aid the development of therapies for the treatment of stress-related disorders.


Assuntos
Núcleo Dorsal da Rafe/fisiologia , Vias Neurais/fisiologia , Neurônios Serotoninérgicos/fisiologia , Estresse Psicológico/fisiopatologia , Transmissão Sináptica/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Núcleo Dorsal da Rafe/citologia , Núcleo Dorsal da Rafe/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo
16.
Nat Commun ; 11(1): 4214, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843632

RESUMO

Stomata are epidermal structures that modulate gas exchanges between plants and the atmosphere. The formation of stomata is regulated by multiple developmental and environmental signals, but how these signals are coordinated to control this process remains unclear. Here, we showed that the conserved energy sensor kinase SnRK1 promotes stomatal development under short-day photoperiod or in liquid culture conditions. Mutation of KIN10, the catalytic α-subunit of SnRK1, results in the decreased stomatal index; while overexpression of KIN10 significantly induces stomatal development. KIN10 displays the cell-type-specific subcellular location pattern. The nuclear-localized KIN10 proteins are highly enriched in the stomatal lineage cells to phosphorylate and stabilize SPEECHLESS, a master regulator of stomatal formation, thereby promoting stomatal development. Our work identifies a module links connecting the energy signaling and stomatal development and reveals that multiple regulatory mechanisms are in place for SnRK1 to modulate stomatal development in response to changing environments.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Estômatos de Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Mutação , Fosforilação , Fotoperíodo , Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
17.
Nat Commun ; 11(1): 4271, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848153

RESUMO

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.


Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Organelas/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento Tridimensional , Microscopia Confocal , Fotodegradação , Imagem com Lapso de Tempo
18.
Zhonghua Yan Ke Za Zhi ; 56(6): 447-455, 2020 Jun 11.
Artigo em Chinês | MEDLINE | ID: mdl-32842327

RESUMO

Objective: To investigate the cellular changes and quantitative analysis of basal cell density (BCD) and corneal epithelial thickness (CET) in limbal stem cell deficiency (LSCD) by in vivo confocal microscopy (IVCM). Methods: Prospective case-control study. A total of 35 eyes of 23 patients diagnosed with LSCD and 25 eyes from normal subjects were included in this study. Based on slit-lamp presentation and the global consensus on classification, the LSCD patients were classified into LSCD Ⅰ, LSCD Ⅱ and LSCD Ⅲ. Confocal images of the central cornea, and the superior, inferior, nasal and temporal limbus were inspected by IVCM. Morphologic characteristics of LSCD were summarized. The BCD and CET in all locations were measured. ANVOA or Kruskal-Wallis test was used for analysis when appropriate. A receiver operating characteristic was used to detect the diagnosis efficiency of BCD and CET. Results: The characteristics in the corneal epithelium of LSCD on IVCM included nested corneal epithelial cells, goblet cells with hyper-reflective spots, irregular basal cells and decreasing subbasal nerve density. The mean BCD of the LSCD group was (8 976±1 096) cells/mm2 in the central cornea. Compared to the control group, the BCD in the central cornea, superior, inferior, nasal and temporal limbus decreased by 30.2%, 26.0%, 28.7%, 29.3% and 30.2%, respectively (all P<0.007). The CET in the central cornea was (47.3±8.1) µm. The CET in the central cornea, superior, inferior, nasal and temporal limbus decreased by 27.9%, 23.7%, 20.6%, 26.9% and 23.1%, respectively, compared to the control group (all P<0.007). There was a decline of BCD and CET in more serious LSCD. Additionally, the decline of BCD and CET was shown in the unaffected region. The receiver operating characteristic showed the diagnosis efficiency of BCD in the corneal center and limbus (0.931 and 0.916) was superior to CET (0.853 and 0.817). Conclusions: There was a series of characteristic cellular changes in LSCD on IVCM. Both BCD and CET decreased significantly in LSCD. The BCD had higher diagnostic efficiency for LSCD.(Chin J Ophthalmol, 2020, 56:447-455).


Assuntos
Doenças da Córnea , Epitélio Anterior/diagnóstico por imagem , Limbo da Córnea , Estudos de Casos e Controles , Humanos , Microscopia Confocal , Estudos Prospectivos , Células-Tronco
19.
Dermatol Online J ; 26(3)2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32609438

RESUMO

Reflectance confocal microscopy (RCM) is a noninvasive real-time imaging technique that has been widely used for the diagnosis of skin cancer. More recently, it has been reported as a useful tool for the diagnosis and management of several inflammatory and infectious skin disorders. This article provides an overview of the current available applications of RCM use in cutaneous infections and infestations. PubMed was used to search the following terms in various combinations: reflectance confocal microscopy, skin, hair, nail, infection, parasitosis, mycosis, virus, bacteria. All papers were accordingly reviewed. In most cutaneous infections or infestations, the main alterations are found in the epidermis and upper dermis, where the accuracy of confocal microscopy is nearly similar to that of histopathology. The high resolution of this technique allows the visualization of most skin parasites, fungi, and a few bacteria. Although viruses cannot be identified because of their small size, viral cytopathic effects can be observed on keratinocytes. In addition, RCM can be used to monitor the response to treatment, thereby reducing unnecessary treatments.


Assuntos
Microscopia Confocal/métodos , Dermatopatias Infecciosas/diagnóstico por imagem , Dermatopatias Parasitárias/diagnóstico por imagem , Feminino , Humanos , Masculino , Pele/diagnóstico por imagem , Pele/microbiologia , Pele/parasitologia , Pele/patologia , Dermatopatias Infecciosas/patologia , Dermatopatias Parasitárias/patologia
20.
Nat Commun ; 11(1): 3281, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612175

RESUMO

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.


Assuntos
Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Febre do Vale de Rift/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Proteínas não Estruturais Virais/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Chlorocebus aethiops , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Agregação Patológica de Proteínas/metabolismo , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/patogenicidade , Células Vero , Proteínas não Estruturais Virais/química , Virulência , Fatores de Virulência
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